1. Field
This disclosure relates to microfiltration. More particularly, the present disclosure describes devices, methods, apparatus, and systems for microfiltration that can be used for performing cell separation and related investigations, such as for the enrichment of viable circulating tumor cells from blood.
2. Description of Related Art
Cell separation has been a challenge for many years especially when the separation is aimed at performance of several investigations in fields such as biochemistry, molecular biology and biotechnology. Cell separation is considered especially important for those investigations performed on cells that express particularly differentiated functions and are desirably separated from an organic or inorganic sample in a viable state. Examples of such investigations include prognostic and diagnostic assays developed in the medical field, herein exemplified by investigation performed on tumor cells, and in particular on circulating tumor cells in metastatic cancer. Metastasis is the spread of cancer from a primary site to non-adjacent secondary sites through a series of sequential steps, which may include the transport of cancer cells through the circulatory system. When tumor cells are transported in the circulatory system, they are called circulating tumor cells (CTCs) in the bloodstream. CTCs are prognostically critical, associated with clinical stage, disease recurrence, tumor metastasis, treatment response, and patient survival following therapy. CTCs may be used as a surrogate and independent marker for assessing the risk of relapse, guiding course of therapy and treatment monitoring for cancer patients.
The majority of the metastatic tumors are derived from epithelial cells, which have unique properties, in comparison to cells of hematogeneous origin. Therefore, separation of CTC from blood constitutes a particularly challenging example of cell separation, since it is performed from a sample where the cells to be separated are comprised together with other cells that although sharing several features with the CTC, are of no interest for the investigation at issue.
Several approaches have been developed to separate CTCs from blood and enrich CTCs from blood, that are based on distinctive features of different cells types. In particular, exploitation of the intrinsic difference between different cell types forms the foundations to the variety of methods for enriching CTCs of epithelial origin from patient blood.
In a first series of approaches, density gradient centrifugation is traditionally employed to enrich the mononucleocyte (MNCs) fraction, which includes CTCs due to their similar buoyant density. The washed MNC fraction cells are cytospun onto glass slides followed by immunofluorescent staining for epithelial markers (predominantly cytokeratin) to detect CTCs. A trained pathologist typically examines each slide for the presence of CTCs. Moreover, density gradient centrifugation typically has a maximum recovery rate of ˜70%.
More recent technologies for CTC enrichment and analysis take advantage of specific surface antigens such as epithelial cell adhesion molecule (EpCAM) for selective capture of epithelial cells. Cellsearch™ is the only FDA approved automated system to capture and assess CTCs to determine the prognosis for metastatic breast, colorectal or prostate cancer. It is based on immunomagnetic separation, in which EpCAM conjugated magnetic beads are used to capture EpCAM positive CTCs from blood under magnetic field. Although clinical studies substantiate its prognostic value, the assay is subject to a large range of recovery rates (9%-90%) due to variable expression of surface markers. An immunoaffinity based “CTC chip” has been developed, where CTCs are captured with antibodies conjugated to surface of micro posts in a microflow chamber. This “CTC chip” has demonstrated CTC capture and detection from patient samples of multiple metastatic cancers. While viable CTCs with high purity can be obtained, the capture efficiency is limited by the variability of surface antigen expression. The throughput and processing time is limited by the stringent conditions of the controlled flow.
Alternatively, techniques for isolation of CTCs based on cell size have been used since the 1960s. Such techniques have been demonstrated to be an efficient, inexpensive and user-friendly way for enrichment of CTCs. Circulating epithelial tumor cells are significantly larger than the surrounding blood cells, where a pore size of ˜8 μm in diameter in the isolating medium has been shown to be optimal for CTC retention. Typically, polycarbonate filters are used as the isolating medium for CTC retention. Such filters are typically fabricated using track etching, which results in random placement of pores with relatively low density that often results in fusion of two or more pores, resulting in a lower CTC capture efficiency of 50-60%. In view of the particularly challenging nature of CTC separation from blood, techniques developed for CTC separation can be applied, with proper adjustments, to separate a wide variety of cell types from a wide variety of samples.